(i) Field of the Invention
The present invention relates to a method of preparing cardiomyocytes selectively and efficiently from ES cells and other pluripotent stem cells, and to cells for regenerative medicine obtained by this method.
(ii) Description of the Related Art
In general, cardiomyocytes undergo active cell division with beating autonomously before birth, but immediately after birth they lose the ability to divide, and since they have little undifferentiated precursor cells, when cardiomyocytes die due to exposure to various forms of stress including myocardial infarction, myocarditis and the like, the lost cardiomyocytes cannot be replaced. As a result, the surviving cardiomyocytes try to maintain myocardial function through compensatory hypertrophy and the like, but if the stress continues and exceeds an allowable threshold, it leads to further exhaustion and death of cardiomyocytes and a consequent lowering of myocardial function (that is, heart failure).
Heart failure and other types of heart disease are the second leading cause of death in Japan, and prognoses are very poor, with a 5-year survival rate of only about 50% for patients with heart diseases. Therefore, it is hoped that development of highly effective therapies for heart failure will lead to great advances in medical welfare as well as improved medical economics. Conventional therapeutic drugs for heart failure include digitalis preparations that increase the contractive force of the myocardium and xanthine preparations and other heart stimulants, but long-term administration of these drugs is known to make the condition worse because there is too much expenditure of myocardial energy. More recently, mainstream therapy has shifted to beta-blockers and ACE inhibitors, which reduce the excess burden on the heart due to stimulation of the sympathetic nervous system and rennin-angiotensin system, but these methods only deal with the immediate symptoms and cannot restore damaged cardiac tissue. By contrast, heart transplantation is a fundamental treatment for severe heart failure, but it is one that is difficult to apply commonly because of such problems as the shortage of heart donors, ethical concerns, the physical and financial burden on patients and the like.
Therefore, it would seem that methods of transplantation to replace weakened or lost cardiomyocytes would be extremely useful for the treatment of heart failure. In fact, it is known from animal experiments that when immature cardiomyocytes obtained from fetuses are transplanted into adult cardiac tissue, the transplanted cells function effectively (See Non-Patent Document 1). However, it is difficult to obtain sufficient cardiomyocytes for this method, and application to clinical medicine is also difficult from an ethical standpoint.
Attention has therefore focused in recent years on inducing differentiation of stem cells into cardiomyocytes and using these cells for transplantation. At present it has not yet been possible to clearly identify a population of precursor cells or stem cells capable of producing cardiomyocytes in adult cardiac tissue, so pluripotent stem cells, which are less differentiated and can differentiate into a variety of cells, are considered to be useful for above method.
Pluripotent stem cells are defined as cells which are capable of indefinite or long-term cell proliferation while remaining in an undifferentiated state in an in vitro culture, which retain normal karyotypes, and which have the ability to differentiate into all of three germ layers (ectoderm, mesoderm and endoderm). At present, the three well-known pluripotent stem cells are embryonic stem cells (ES cells) derived from early-stage embryos, embryonic germ cells (EG cells) derived from primordial germ cells at the embryonic stage, and multipotent adult progenitor cells (MAPC) isolated from adult bone marrow.
It has long been known that ES cells in particular can be induced to differentiate into cardiomyocytes in vitro. Most of the early studies were done by using ES cells derived from mice. When ES cells are cultured in floating culture as single cells (individual cells dispersed with no adhesion between cells due to enzyme treatment or the like) without the presence of a differentiation-inhibiting factor such as leukemia inhibitory factor (LIF) or the like, the ES cells adhere to one another and aggregate, forming a structure called embryoid bodies (EBs) which are similar to the early embryonal structures. It is also known that cardiomyocytes with spontaneous beating ability appear when these EBs are cultured in suspension or in adhesion on the surface of culture devices.
ES cell-derived cardiomyocytes prepared as described above exhibit very similar properties to those of immature cardiomyocytes in fetal hearts (See Non-Patent Documents 2 and 3). Moreover, it has been confirmed from animal experiments that when ES cell-derived cardiomyocytes are transplanted into adult cardiac tissues they are highly effective, with results similar to those obtained by transplantation of fetal myocardium (See Patent Document 1; Non-Patent Document 4).
In 1995, Thomson et al first established ES cells from primates (See Patent Document 2; Non-Patent Document 5), and thus the regeneration therapy using pluripotent stem cells-derived cardiomyocytes has become realistic. Subsequently they also succeeded in isolating and establishing human ES cell lines from early human embryos (See Non-Patent Document 6). Moreover, Gearhart et al established human EG cell lines from primordial human germ cells (See Non-Patent Document 7; Patent Document 3). Kehat et al (See Non-Patent Document 8) and Xu et al (See Patent Document 4; Non-Patent Document 9) have reported that human ES cells can differentiate into cardiomyocytes in vitro, as mouse ES cells can do. According to these reports, human ES cells-derived cardiomyocytes which have been induced to differentiate from human ES cells not only have the ability to beat spontaneously but also express and produce myocardial-specific proteins such as myosin heavy and light chains, alpha-actinin, troponin I and atrial natriuretic peptide (ANP) and myocardial-specific transcription factors such as GATA-4, Nkx2.5, MEF-2c and the like, and from microanatomical observation and electrophysiological analysis it appears that they retain the properties of immature cardiomyocytes at the fetal stage, and could be used for regenerative therapy.
However, one serious problem remains to be elucidated to use pluripotent stem cells-derived cardiomyocytes for cell transplantation therapy and other purposes. When EBs are formed from ES cells or EG cells by conventional methods, not only cardiomyocytes, but also other types of differentiated cells, such as blood cells, vascular cells, neural cells, intestinal cells, bone and cartilage cells and the like, are developed. Moreover, the proportion of cardiomyocytes in these differentiated cell population is not so high, only about 5 to 20% of the total.
Methods of isolating only cardiomyocytes from a mixture of various kinds of cells include a method of adding an artificial modification to the ES cell genes, conferring drug resistance or ectopic expression, and collecting cells having the properties of cardiomyocytes or precursor cells thereof. For example, by introducing a gene cassette capable of expressing a neomycin (G418) resistance gene under the control of the α-myosin heavy chain promoter into mouse ES cells, Field and his co-researchers established a system in which those ES cells, could only survive in medium to which G418 had been added when they differentiated into cardiomyocytes and expressed the α-myosin heavy chain gene (See Patent Document 1; Non-Patent Document 4). 99% or more of G418-resistant cells selected by this method were confirmed to be cardiomyocytes. However, although the purity of the cardiomyocytes is extremely high in this method the final number of cardiomyocytes obtained is only a few percent of the total cell count, making it difficult to obtain enough amounts of cardiomyocytes for transplantation.
Recently, Chunhui et al have reported that when human ES cells are treated with 5-azacytidine, the percentage of troponin I-positive cells (candidate cardiomyocytes) in EBs rises from 15% to 44% (See Non-Patent Document 9), but even in this method the percentage of cardiomyocytes in EBs does not exceed 50%. Moreover, 5-azacytidine is a demethylation agent that alters the expression of genes by removing methyl groups bound to DNA, and because it acts directly on the chromosomes, it is not a suitable drug for preparing cells for cell transplantation.
Other methods for producing cardiomyocytes more efficiently from ES cells include, in the case of mouse ES cells, addition of retinoic acid (See Non-Patent Document 10), ascorbic acid (See Non-Patent Document 11), TGF beta, BMP-2 (See Non-Patent Document 12), PDGF (See Non-Patent Document 13) and Dynorphin B (See Non-Patent Document 14) and treatment to increase reactive oxygen species (ROS) (See Non-Patent Document 15) and Ca2+ (See Non-Patent Document 16) in the cells, all of which are known to act positively to induce cardiomyocyte differentiation. However, cardiomyocyte-specific, selective differentiation has not been achieved with any of these methods.
The secretory proteins Noggin and Chordin were initially identified as nerve induction factors in Xenopus embryos (See Non-Patent Documents 17 and 18; Patent Documents 5-8). Further study has shown that Noggin and Chordin bind with BMP (Bone Morphogenic Protein) family of molecules, which impair the signal transduction, and cause neural induction and differentiation. (See Non-Patent Documents 19-21). In fact, experiments using mouse ES cells have shown that nerve cell differentiation is induced in cells in which the Noggin or Chordin gene is constantly expressed (See Non-Patent Document 22).
When human ES cells are cultured in medium to which Noggin has been added, the function of endogenously produced BMP-2 is diminished, and ES cells into extraembryonic endodermal cells are impaired and so that they are maintained in an undifferentiated state. In addition, when noggin-treated ES cells are subsequently cultured under neural differentiation condition, the development of neural cells is induced (See Patent Document 9).
It has also been reported from earlier studies using chicken (See Non-Patent Document 23), Xenopus (See Non-Patent Document 24) and mouse embryonic carcinoma cells (See Non-Patent Document 25) that the BMP family of molecules acts to promote development and/or differentiation of cardiomyocytes, and when that action is blocked by Noggin treatment, development and/or differentiation of cardiomyocytes is suppressed.
Heretofore there have been no efforts to encourage development and differentiation of cardiomyocytes by using Noggin, Chordin or other BMP signal-inhibiting factors.    Patent Document 1: U.S. Pat. No. 6,015,671;    Patent Document 2: U.S. Pat. No. 5,843,780;    Patent Document 3: U.S. Pat. No. 6,090,622;    Patent Document 4: International Patent Disclosure 03/06950, pamphlet;    Patent Document 5: International Patent Disclosure 94/05791, pamphlet;    Patent Document 6: U.S. Pat. No. 5,679,783;    Patent Document 7: U.S. Pat. No. 5,846,770;    Patent Document 8: U.S. Pat. No. 5,986,056;    Patent Document 9: International Patent Disclosure 01/98463, pamphlet;    Non-Patent Document 1: Soonpaa et al, Science 264:98, 1994;    Non-Patent Document 2: Maltsev et al, Mech. Dev. 44:41, 1993;    Non-Patent Document 3: Maltsev et al, Circ. Res. 75: 233, 1994    Non-Patent Document 4: Klug et al, J. Clin. Invest. 98: 216, 1996;    Non-Patent Document 5: Thomson et al, Proc. Natl. Acad. Sci. USA 92:7844, 1995;    Non-Patent Document 6: Thomson et al, Science 282: 114, 1998;    Non-Patent Document 7: Shamblott et al, Proc. Natl. Acad. Sci. USA 95: 13726, 1998;    Non-Patent Document 8: Kehat et al, J. Clin. Invest. 108: 407, 2001;    Non-Patent Document 9: Xu et al. Circ. Res. 91: 501, 2002;    Non-Patent Document 10: Wobus et al, J. Mol. Cell. Cardiol. 29: 1525, 1997;    Non-Patent Document 11: Takahashi et al, Circulation 107: 1912, 2003;    Non-Patent Document 12: Behfar et. al, FASEB J. 16: 1558, 2002;    Non-Patent Document 13: Sachinidis et al, Cardiovasc. Res. 58: 278, 2003;    Non-Patent Document 14: Ventura et al, Circ. Res. 92: 623, 2003;    Non-Patent Document 15: Sauer et al, FEBS Lett. 476:218, 2000;    Non-Patent Document 16: Li et al, J. Cell Biol. 158: 103, 2002;    Non-Patent Document 17: Smith & Harland, Cell 70: 829, 1992;    Non-Patent Document 18: Sasai et al, Cell 79: 779, 1994;    Non-Patent Document 19: Re'em-Kalma et al, Proc. Natl. Acad. Sci. USA 92: 12141, 1995;    Non-Patent Document 20: Zimmerman et al, Cell 86: 599, 1996;    Non-Patent Document 21: Piccolo et al, Cell 86: 589, 1996;    Non-Patent Document 22: Gratsch & O'Shea, Dev. Biol. 245: 83, 2002;    Non-Patent Document 23: Schultheiss et al, Genes Dev., 11: 451, 1997;    Non-Patent Document 24: Sparrow et al, Mech. Dev. 71:151, 1998    Non-Patent Document 25: Monzen et al, Mol. Cell. Biol. 19:7096, 1999.